Given the poor track record to date of animal models for creating cardioprotective drugs, human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) have been proposed as a therapeutically relevant human platform to guide target validation and cardiac drug development. Mitogen-Activated Protein Kinase Kinase Kinase Kinase-4 (MAP4K4) is an "upstream" member of the MAPK superfamily that is implicated in human cardiac muscle cell death from oxidative stress, based on gene silencing and pharmacological inhibition in hPSC-CMs. A further role for MAP4K4 was proposed in heart muscle cell death triggered by cardiotoxic anti-cancer drugs, given its reported activation in failing human hearts with doxorubicin (DOX) cardiomyopathy, and its activation acutely by DOX in cultured cardiomyocytes. Here, we report successful protection from DOX in two independent hPSC-CM lines, using two potent, highly selective MAP4K4 inhibitors. The MAP4K4 inhibitors enhanced viability and reduced apoptosis at otherwise lethal concentrations of DOX, and preserved cardiomyocyte function, as measured by spontaneous calcium transients, at sub-maximal ones. Notably, in contrast, no intereference was seen in tumor cell killing, caspase activation, or mitochondrial membrane dissipation by DOX, in human cancer cell lines. Thus, MAP4K4 is a plausible, tractable, selective therapeutic target in DOX-induced human heart muscle cell death. With earlier diagnosis and improved treatments for cancer, cardiovascular disease has become the main alternative cause of death in cancer survivors 1-4. This heightened risk is ascribable to the cardiac toxicity of several routine anti-cancer agents including, especially, anthracyclines like doxorubicin (DOX) 5. Though some relief can result from standard heart failure medications, no approved therapy apart from the iron chelator dexrazoxane addresses the responsible cytotoxic mechanisms and effector pathways operating in the damaged cardiomyocytes. For anthracyclines, these include diverse reported mediators-binding to nuclear topoisomerase 2β, thereby triggering DNA double-strand breaks, p53-dependent apoptosis, mitochondrial dysfunction, reactive oxygen species, and programmed iron-dependent cell death (ferroptosis) 1,6,7. Cardiotoxicity can be acute, early, or late; early intervention based on subclinical abnormalities may be key to averting the delayed or cumulative effects 8. Notably, acute toxicity is seen in up to 30% of patients receiving anthracyclines, soon after infusion 4. Current cardioprotection trials in cancer rely chiefly on routine heart failure medications (ACE inhibitors, β-blockers, angiotensin receptor blockers), which mitigate the symptoms and signs, with little or no demonstrated impact on cardiomyocyte death. Therapeutic progress has been hampered, in part, by the failure of animal models alone to predict clinical success in cardioprotection. For instance, nearly all strategies for protection in ischemic heart disease, a more intensively studied indication, have failed between phases I and I...
Resistance to Immune Checkpoint Blockade (ICB) constitutes the current limiting factor for the optimal implementation of this novel therapy, which otherwise demonstrates durable responses with acceptable toxicity scores. This limitation is exacerbated by a lack of robust biomarkers. In this study, we have dissected the basal TME composition at the gene expression and cellular levels that predict response to Nivolumab and prognosis. BCR, TCR and HLA profiling were employed for further characterization of the molecular variables associated with response. The findings were validated using a single-cell RNA-seq data of metastatic melanoma patients treated with ICB, and by multispectral immunofluorescence. Finally, machine learning was employed to construct a prediction algorithm that was validated across eight metastatic melanoma cohorts treated with ICB. Using this strategy, we have unmasked a major role played by basal intratumoral Plasma cells expressing high levels of IGKC in efficacy. IGKC, differentially expressed in good responders, was also identified within the Top response-related BCR clonotypes, together with IGK variants. These results were validated at gene, cellular and protein levels; CD138+ Plasma-like and Plasma cells were more abundant in good responders and correlated with the same RNA-seq-defined fraction. Finally, we generated a 15-gene prediction model that outperformed the current reference score in eight ICB-treated metastatic melanoma cohorts. The evidenced major contribution of basal intratumoral IGKC and Plasma cells in good response and outcome in ICB in metastatic melanoma is a groundbreaking finding in the field beyond the role of T lymphocytes.
In order to identify cellular phenotypes resulting from nonsense (gain of stop/premature termination codon) variants, we devised a framework of analytic methods that minimised confounder contributions, and applied to blood outgrowth endothelial cells (BOECs) derived from controls and patients with heterozygous nonsense variants in ACVRL1, ENG or SMAD4 causing hereditary haemorrhagic telangiectasia (HHT). Following validation of 48 pre-selected genes by single cell qRT-PCR, discovery RNASeq ranked HHT-differential alignments of 16,807 Ensembl transcripts. Consistent gene ontology (GO) processes enriched compared to randomly-selected gene lists included bone morphogenetic protein, transforming growth factor-β and angiogenesis GO processes already implicated in HHT, further validating methodologies. Additional terms/genes including for endoplasmic reticulum stress could be attributed to a generic process of inefficient nonsense mediated decay (NMD). NMD efficiency ranged from 78-92% (mean 87%) in different BOEC cultures, with misprocessed mutant protein production confirmed by pulse chase experiments. Genes in HHT-specific and generic nonsense decay (ND) lists displayed differing expression profiles in normal endothelial cells exposed to an additional stress of exogenous 10μmol/L iron which acutely upregulates multiple mRNAs: Despite differing donors and endothelial cell types, >50% of iron-induced variability could be explained by the magnitude of transcript downregulation in HHT BOECs with less efficient NMD. The Genotype Tissue Expression (GTEx) Project indicated ND list genes were usually most highly expressed in non-endothelial tissues. However, across 5 major tissues, although 18/486 nonsense and frameshift variants in highly expressed genes were captured in GTEx, none were sufficiently prevalent to obtain genome-wide significant p values for expression quantitative trait loci (GnomAD allele frequencies <0.0005). In conclusion, RNASeq analytics of rare genotype-selected, patient-derived endothelial cells facilitated identification of natural disease-specific and more generic transcriptional signatures. Future studies should evaluate wider relevance and whether injury from external agents is augmented in cells with already high burdens of defective protein production.
1285 Identifying the key regulatory genes that direct human ES/iPS cell differentiation to certain lineages is required before considering these cells for their potential clinical application. It has been proposed that the cellular starting material used to generate novel iPS cell lines imparts an epigenetic memory which in turn influences the iPS lines' potential to differentiate towards certain developmental lineages. To determine novel key regulators that direct ES/iPS cell differentiation to hematopoietic lineages, we decided to compare differentiation capacity of multiple iPS cell lines generated from two neonatal mesodermal tissue derived cell types. For this, we generated multiple iPS cell lines from umbilical cord blood derived endothelial cells and amniotic fluid derived mesenchymal stem cells, using an identical dedifferentiation system/protocol. Despite the similar germ layer origin of these iPS derived cell lines, these lines showed significant differences in blood generation efficiency and lineage specification capacity. From the 8 cord blood derived iPS cell lines we tested, the average efficiency of hematopoietic (CD45+) cell generation was 34.6+/−21.2% (range 9.8 to 65.8%), of which 5.8+/− 4% were CD45+CD34+ hematopoietic progenitors. From the 9 mesenchymal stem cell derived iPS lines, the average efficiency of CD45+ hematopoietic cells was 14.2+/− 9% (range 1.6 to 26.3%), of which 2.9+/− 1.9% were CD45+CD34+ hematopoietic progenitors (Figure 1A). Note, using our standardized iPS-2-Blood differentiation protocol the efficiency of blood differentiation for individual iPS cell lines was highly reproducible. These results indicate a significant increase in the efficiency of blood generation from the endothelial derived iPS cell lines compared to the mesenchymal stem cell derived iPS cell lines, and suggest that the close ontological relationship between hematopoietic cells and endothelial cells results in the increased efficiency of blood generation seen here. We further compared the iPS cell lines for their potential to differentiate into hematopoietic cells of the myeloid, erythroid, and lymphoid lineages. Both mesoderm lineage derived iPS cell lines showed myeloid and lymphoid lineage differentiation potential as evidenced by CFU assay for presence of macrophages and granulocytes, and by 4-week OP9, and OP9-DL1 culture assays showing CD19, CD10, CD34 expression for B cells, CD1a, CD7, CD5, CD4, TCRb, CD3 expression for T cells, and CD56, CD16 for NK cells. However, further comparative analysis revealed a limited erythroid (BFU-E) potential of cord blood derived iPS cell lines compared to amniotic fluid derived iPS cell lines (Figure 1B). Most amniotic fluid derived iPS lines showed significantly erythroid cell potential, where as the cord blood derived iPS lines showed significantly reduced or no erythroid cell potential. These results suggest that the endothelial origin of cord blood derived iPS lines imparts an epigenetic memory that results in the reduced efficiency of erythroid cell generation compared with mesenchymal stem cell derived iPS lines. The difference in the efficiency of hematopoietic cell generation and lineage specification between the cord blood and amniotic fluid derived iPS lines suggests that iPS reprogramming to a pluripotent state is not complete (despite iPS cell line validation of pluripotency via teratocarcinoma assay), and significant gene expression differences exist between the lines. We are currently performing gene expression and epigenetic profile analyses of these closely related mesodermal lines, and expect that the close ontological relationship of the two mesoderm derived cell types will facilitate the deconvolution of the molecular basis of the differences.Figure 1.Differences in blood differentiation efficiency and lineage potential of iPS lines derived from two ontologically related cell types.Figure 1. Differences in blood differentiation efficiency and lineage potential of iPS lines derived from two ontologically related cell types. Disclosures: No relevant conflicts of interest to declare.
No abstract
Retinoic acid (RA) is a key morphogen involved in the establishment of various cell-fates in the developing embryo. The establishment of certain tissues, including mesodermal derivates such as the somites and heart depends on restricted exposure to RA during embryonic development, however, in regard to the possible involvement of RA for the emergence of blood in the human embryo very little is known. By using an inhibitor of cellular RA synthesis (DEAB) together with our human iPS-to-blood differentiation system we report enhanced generation of human hematopoietic progenitors cells (CD43/45+,CD34+), and early progenitor cells with an adult hematopoietic stem cell phenotype (CD43/45+,CD34+,CD38,CD90+,CD45RA-) possessing myeloid and lymphoid differentiation capacity. RA inhibition increased the output of these early progenitor cells by 2.7-fold (p-value: 0,006, n=6) compared to DMSO control, and increased the number of clonogenic progenitors (CFUs) by 2-fold (p-value: 0,022, n=6). This improvement is consistent when using both a human ES line (Hues3) and a human iPS-line (RB9-CB1, generated from cord blood). RA inhibition also increased the number of iPS-derived blood progenitors capable of differentiating into the lymphoid lineage, generating phenotypic T-lineage and natural killer cells after sub-culture on OP9-DL1 stroma. Conversely, and in support of our findings, directly adding RA was found to severely decrease the blood generation efficiency, with the early progenitor fraction significantly decreased even at low levels of RA exposure. In order to assess how the RA inhibition led to the increase in early progenitors, whether by directing pluripotent stem cell differentiation of mesodermal precursors towards blood, or by inferring an early progenitor cell maintenance and/or expansion, we performed the following analyses. Firstly, the loss of clonogenic progenitors was reduced 1.6 fold with extended culture in the presence of the inhibitor. Secondly, within the iPS derived blood (CD43/45+) fraction of the cultures, RA inhibition increased the frequency of the CD34+,CD38-,CD90+,CD45RA- compartment by 2.1-fold (p-value: 0,021, n=6). Similarly when comparing the CFU-forming capacity from equal numbers of sorted hematopoietic progenitors (CD43/45+,CD34+), cells generated with the inhibitor demonstrated 2-fold higher CFU frequency suggesting increased preservation of CFU-forming cells within the CD34+ progenitor fraction. Together this suggests that progenitor maintenance is improved with the inhibitor, however, the contribution to the total fold expansion seen with RA inhibition cannot be attributed solely to the increased maintenance of early progenitor cells in the culture. Using multi-timepoint RT-qPCR of samples obtained during the differentiation protocol, we analysed expression of key developmental genes and saw improved commitment of the differentiating culture towards mesoderm (BRACHYURY), decreased amounts of anterior lateral plate/cardiogenic mesoderm (NKX2.5), and enhanced expression of genes known to be relevant for definitive hematopoiesis and hemogenic endothelium (RUNX1, FLK1, APLNR, PDGFRa). In addition, preliminary data show that RA inhibition increases the frequency of the CD34hi,CD90hi,CD43neg population observed at day 8, recently described as the precursor population of definitive hematopoiesis, indicating that RA inhibition confers an early positive effect on the developmental commitment towards the hematopoietic mesoderm. Together, our data indicate that modulation of retinoic acid signaling improves blood generation from human pluripotent stem cell lines both by improving the development towards the hematopoietic mesoderm by favoring mesodermal blood precursor commitment, and by preserving the immature progenitor population from differentiation. Therefore, we propose that RA is a negative regulator of mesodermal formation and the specification towards hematopoietic precursors, and that it promotes the exhaustion of definitive hematopoietic progenitors. This suggests the possibility that blood evolutionarily formed in a region of the mesoderm where RA exposure from neighboring tissues, such as RA producing ectoderm, was restricted. Therefore, using small molecules to inhibit RA signaling is a novel approach to improve the generation of human blood in vitro. Disclosures: Rönn: Primorigen Biosciences: Co-inventor on pending patent. No financial ties., Co-inventor on pending patent. No financial ties. Patents & Royalties. Guibentif:Primorigen Biosciences: Co-inventor on pending patent. No financial ties., Co-inventor on pending patent. No financial ties. Patents & Royalties. Garcia:Primorigen Biosciences: Employment. Woods:Primorigen Biosciences: Co-inventor on pending patent., Co-inventor on pending patent. Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees.
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